The Experts below are selected from a list of 117 Experts worldwide ranked by ideXlab platform
Mikio Nishimura - One of the best experts on this subject based on the ideXlab platform.
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microbody defective mutants of arabidopsis
Journal of Plant Research, 1998Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Kanako Toriyama, Shoji Mano, Katsushi Yamaguchi, Maki Kondo, Hiroshi HayashiAbstract:In germinating fatty seedlings, Microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of Microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of Microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described.
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hydroxypyruvate reductase with a carboxy terminal targeting signal to Microbodies is expressed in arabidopsis
Plant and Cell Physiology, 1997Co-Authors: Shoji Mano, Makoto Hayashi, Maki Kondo, Mikio NishimuraAbstract:Five Arabidopsis EST cDNA clones of hydroxypyruvate reductase (HPR), a photorespiratory enzyme in leaf peroxisomes, were sequenced. Deduced amino acid sequences revealed that HPR in Arabidopsis contained the carboxy-terminal targeting signal to Microbodies. Nucleotide sequence analysis showed that the cDNA with the longest insert contained an open reading frame of 1,158 bp which encoded a polypeptide with 386 amino acids with a calculated molecular mass of 42,251 Da. A Southern blot analysis suggested that the Arabidopsis HPR gene, like that of the pumpkin HPR gene, exists as a single copy. Two kinds of pumpkin HPR mRNA might be produced from a single gene by alternative splicing, but the structure of the genomic DNA indicated that the Arabidopsis HPR gene did not undergo alternative splicing. We detected a polypeptide with a molecular mass of 42 kDa in green leaves of Arabidopsis using an HPR-specific antibody. Immunoelectron microscopy revealed that Arabidopsis HPR protein was exclusively localized in leaf peroxisomes in green leaves. These results indicate that HPR is expressed in a form with a carboxy-terminal targeting signal to Microbodies and is localized in Microbodies in Arabidopsis, suggesting that the differences in the gene structure and the regulation of gene expression of HPR are probably due to species-specific differences in plants.
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changes in targeting efficiencies of proteins to plant Microbodies caused by amino acid substitutions in the carboxy terminal tripeptide
Plant and Cell Physiology, 1997Co-Authors: Makoto Hayashi, Maki Kondo, Masahiro Aoki, Mikio NishimuraAbstract:It has been demonstrated that the carboxyl terminus of microbody enzymes functions as a targeting signal to Microbodies in higher plants. We have examined an ability of 24 carboxy-terminal amino acid sequences to facilitate the transport of a cytosolic passenger protein, /^glucuronidase, into Microbodies in green cotyledonary cells of transgenic Arabidopsis. Immunoelectron microscopic analysis revealed that carboxy-terminal tripeptide sequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as a microbodytargeting signal, although tripeptides with proline at the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies. All known microbody enzymes that are synthesized in a form similar in size to the mature molecule, except catalase, contain one of these tripeptide sequences at their carboxyl terminus.
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cdna cloning and expression of a gene for 3 ketoacyl coa thiolase in pumpkin cotyledons
Plant Molecular Biology, 1996Co-Authors: Mikio Nishimura, Yuka Takeuchi, Makoto Hayashi, Akira KatoAbstract:A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a λgt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to Microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of Microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of Microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.
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Functional Transformation of Microbodies in Higher Plant Cells
Cell structure and function, 1996Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Katsushi Yamaguchi, Shoji ManoAbstract:In germinating fatty seedlings, Microbodies are functionally transformed to leaf peroxisomes from glyoxysomes during greening, and then converted to glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to glyoxysomes during senescence. The reversible transformations of Microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.
Marten Veenhuis - One of the best experts on this subject based on the ideXlab platform.
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Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells
Functional & Integrative Genomics, 2009Co-Authors: Jan A. K. W. Kiel, Marco A. Berg, Fabrizia Fusetti, Bert Poolman, Marten Veenhuis, Ida J. KleiAbstract:In the filamentous fungus Penicillium chrysogenum , Microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum Microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum Microbodies.
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A Eukaryote without Catalase-Containing Microbodies: Neurospora crassa Exhibits a Unique Cellular Distribution of Its Four Catalases†
2006Co-Authors: Wolfgang Schliebs, Marten Veenhuis, Wolf-hubert Kunau, Christian Würtz, Hanspeter RottensteinerAbstract:Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing Microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3�-diaminobenzidine and H 2O 2 did not lead to catalasedependent reaction products within Microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to Microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within Microbodies. Microbodies are nearly ubiquitous organelles of the eukaryoti
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proliferation and function of Microbodies in the nematophagous fungus arthrobotrys oligospora during growth on oleic acid or d alanine as the sole carbon source
Fems Microbiology Letters, 1993Co-Authors: Jan Dijksterhuis, Willem Harder, Marten VeenhuisAbstract:The nematophagous fungus Arthrobotrys oligospora is able to grow on oleic acid or d-alanine as the sole carbon source. During growth on oleic acid, activities of enzymes of the β-oxidation pathway, but not catalase, were induced. In the presence of d-alanine, both d-amino acid oxidase and catalase activities were enhanced. Biochemically and cytochemically, the activities of the above enzymes were assigned to Microbodies. The significance of these results in relation to the function of Microbodies in trophic hyphae, which are formed during nematode infection, is discussed.
Makoto Hayashi - One of the best experts on this subject based on the ideXlab platform.
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microbody defective mutants of arabidopsis
Journal of Plant Research, 1998Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Kanako Toriyama, Shoji Mano, Katsushi Yamaguchi, Maki Kondo, Hiroshi HayashiAbstract:In germinating fatty seedlings, Microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of Microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of Microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described.
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hydroxypyruvate reductase with a carboxy terminal targeting signal to Microbodies is expressed in arabidopsis
Plant and Cell Physiology, 1997Co-Authors: Shoji Mano, Makoto Hayashi, Maki Kondo, Mikio NishimuraAbstract:Five Arabidopsis EST cDNA clones of hydroxypyruvate reductase (HPR), a photorespiratory enzyme in leaf peroxisomes, were sequenced. Deduced amino acid sequences revealed that HPR in Arabidopsis contained the carboxy-terminal targeting signal to Microbodies. Nucleotide sequence analysis showed that the cDNA with the longest insert contained an open reading frame of 1,158 bp which encoded a polypeptide with 386 amino acids with a calculated molecular mass of 42,251 Da. A Southern blot analysis suggested that the Arabidopsis HPR gene, like that of the pumpkin HPR gene, exists as a single copy. Two kinds of pumpkin HPR mRNA might be produced from a single gene by alternative splicing, but the structure of the genomic DNA indicated that the Arabidopsis HPR gene did not undergo alternative splicing. We detected a polypeptide with a molecular mass of 42 kDa in green leaves of Arabidopsis using an HPR-specific antibody. Immunoelectron microscopy revealed that Arabidopsis HPR protein was exclusively localized in leaf peroxisomes in green leaves. These results indicate that HPR is expressed in a form with a carboxy-terminal targeting signal to Microbodies and is localized in Microbodies in Arabidopsis, suggesting that the differences in the gene structure and the regulation of gene expression of HPR are probably due to species-specific differences in plants.
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changes in targeting efficiencies of proteins to plant Microbodies caused by amino acid substitutions in the carboxy terminal tripeptide
Plant and Cell Physiology, 1997Co-Authors: Makoto Hayashi, Maki Kondo, Masahiro Aoki, Mikio NishimuraAbstract:It has been demonstrated that the carboxyl terminus of microbody enzymes functions as a targeting signal to Microbodies in higher plants. We have examined an ability of 24 carboxy-terminal amino acid sequences to facilitate the transport of a cytosolic passenger protein, /^glucuronidase, into Microbodies in green cotyledonary cells of transgenic Arabidopsis. Immunoelectron microscopic analysis revealed that carboxy-terminal tripeptide sequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as a microbodytargeting signal, although tripeptides with proline at the first amino acid position and isoleucine at the carboxyl terminus show weak targeting efficiencies. All known microbody enzymes that are synthesized in a form similar in size to the mature molecule, except catalase, contain one of these tripeptide sequences at their carboxyl terminus.
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cdna cloning and expression of a gene for 3 ketoacyl coa thiolase in pumpkin cotyledons
Plant Molecular Biology, 1996Co-Authors: Mikio Nishimura, Yuka Takeuchi, Makoto Hayashi, Akira KatoAbstract:A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a λgt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to Microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of Microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of Microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.
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Functional Transformation of Microbodies in Higher Plant Cells
Cell structure and function, 1996Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Katsushi Yamaguchi, Shoji ManoAbstract:In germinating fatty seedlings, Microbodies are functionally transformed to leaf peroxisomes from glyoxysomes during greening, and then converted to glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to glyoxysomes during senescence. The reversible transformations of Microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.
Akira Kato - One of the best experts on this subject based on the ideXlab platform.
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microbody defective mutants of arabidopsis
Journal of Plant Research, 1998Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Kanako Toriyama, Shoji Mano, Katsushi Yamaguchi, Maki Kondo, Hiroshi HayashiAbstract:In germinating fatty seedlings, Microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of Microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of Microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described.
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cdna cloning and expression of a gene for 3 ketoacyl coa thiolase in pumpkin cotyledons
Plant Molecular Biology, 1996Co-Authors: Mikio Nishimura, Yuka Takeuchi, Makoto Hayashi, Akira KatoAbstract:A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a λgt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to Microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of Microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of Microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.
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Functional Transformation of Microbodies in Higher Plant Cells
Cell structure and function, 1996Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Katsushi Yamaguchi, Shoji ManoAbstract:In germinating fatty seedlings, Microbodies are functionally transformed to leaf peroxisomes from glyoxysomes during greening, and then converted to glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to glyoxysomes during senescence. The reversible transformations of Microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.
Arie J. Verkleij - One of the best experts on this subject based on the ideXlab platform.
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Enrichment of Penicillium chrysogenum Microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.
Biochimica et biophysica acta, 1995Co-Authors: Wally H. Müller, Jeroen Essers, Bruno M. Humbel, Arie J. VerkleijAbstract:Abstract A procedure to enrich Microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the Microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10 000 × g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14 000 × g pellet. After 2 h, 45 000 × g isopycnic centrifugation of the 14 000 × g pellet on a continuous 20–60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing Microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-Microbodies are enriched in the 38% nycodenz fraction. The Microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chrysogenum Microbodies was 1.20 g ml−1 as deduced from the 38% (w/v) nycodenz concentration.
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Involvement of Microbodies in penicillin biosynthesis.
Biochimica et biophysica acta, 1992Co-Authors: Wally H. Müller, Roelof A. L. Bovenberg, Marloes H. Groothuis, Fred Kattevilder, Erik B. Smaal, Lucia H. M. Van Der Voort, Arie J. VerkleijAbstract:Penicillium chrysogenum strains were constructed which express a mutant acyltransferase lacking the putative targeting signal for microbody proteins. The mutated enzyme was located in vacuoles and in neighbouring cytoplasm. Although acyltransferase was expressed in vivo and was active in vitro, the mutants did not produce penicillin. The results demonstrate the involvement of Microbodies in penicillin production.